DNA Technologies – Southern Blotting & ELISA By C. Kohn, Waterford WI

Review  If we wanted to read a gene or even our entire genome base by base (letter by letter), we would use the Sanger Method  The Sanger Method used ddNTPs to stop the same sequence at different places; the ddNTPs would give a color to the last base added

Review  If we wanted to compare someone’s DNA fingerprint, we would use PCR Electrophoresis and Restriction Fragment Analysis  We would cut out a gene, replicate it billions of times, and then cut it up with a restriction fragment  We would then pull the cut up fragments through a gel and compare banding patterns

Overview  The Sanger Method is useful if we want to read large amounts of DNA and know the exact coding sequence of bases  However, the Sanger method is not cheap nor is it as quick. It takes a lot of sophisticated equipment and highly trained individuals to run this test  PCR Electrophoresis and Restriction Fragment Analysis are quick, cheap tests that most people with a little training could perform.  However, the results aren’t as precise as the Sanger Method; we can’t read the actual DNA  We can only compare different kinds of DNA

Southern Blotting  The Southern Blotting test enables us to detect if a specific gene exists on a PCR Electrophoresis DNA fingerprint.  For example, if you wanted to know if you were a carrier for a recessive genetic disease, you could run the PCR Electrophoresis on the section of your DNA that would contain the gene for this disease.  You could then run a Southern Blot on the gel to see if the gene for the disease is actually there

Southern Blotting  In a nutshell, Southern Blotting allows us to determine if a sample of DNA has a specific gene without having to go through the difficulty or expense of running the Sanger Method Test.  It provides us the information of the Sanger Method at the cost and ease of PCR

Southern Blotting Steps Southern Blotting has a few steps.  1. Run PCR Electrophoresis (amplification, addition of a restriction fragment, gel electrophoresis)  2. Transfer the DNA to a membrane  3. Add a probe with the complementary sequence for the gene you are looking for  (e.g. GATCA for CTAGT)  4. If the gene your are looking for is present, the probe will bind to it and create a signal (e.g. it may glow with bioluminescence if the gene is there)

Genes and Disease  Faulty genes regularly cause disease.  A genetic test can easily determine if an individual or animal is a carrier of a genetic disease  This can be especially valuable if the disease is recessive, or if the individual carries but does not express the disease.  (e.g. if they are Rr for a recessive disease, they would carry it but not have the symptoms of it).  Southern Blotting enables us to see if an individual has genes for any kind of genetic disorder.

But what if…  However, not all diseases are genetic  Some diseases are caused by organisms outside of the body  An infectious disease is a disease passed on to us or an animal by another organism  For example, you can get a rhinovirus (cold virus) from another individual. This has nothing to do with your DNA  Would PCR & Southern Blotting be useful in this case?  No, because genetic tests only look at DNA

ELISA  ELISA is a test that checks for proteins instead of DNA.  Proteins are made from information encoded in DNA, but proteins and DNA are two separate things  ELISA detects antibodies in blood  Antibodies are proteins used to ‘label’ things in the blood.  For example, your blood as antibodies that tell your body whether or not you have type A, B, O, or AB blood.  Antibodies are proteins, not genes or DNA

Antibodies  Molecular Post-Its  Antibodies help your body to recognize the antigens for other substances, including diseases.  For example, chicken pox has chicken pox antigens.  Your body would produce chicken pox antibodies to identify the chicken pox antigens.  Antigen is short for “antibody generator”

Antibodies & Antigens  Antibodies and Antigens are like locks and keys  With a lock, only one kind of key will fit  The same is true for antigens and antibodies  The shape of an antibody is specific to the antigen it binds to  An antibody will only bind to one kind of antigen  If an individual has a disease, their blood should produce antibodies specific to that disease.  These antibodies will tell us if you have the disease or not

ELISA Testing – A Test for Antibodies  ELISA works by coating wells (or depressions) in a dish with the antigen proteins for a specific disease  A sample of blood is added to the wells of the dish  If the disease is there, they should have those antibodies in their blood  These antibodies will bind to the antigen in the well  A second round of antigens are added, and these second antigens are dyed a specific color.  If the protein is there, the colored antigen will change the color of the well.

ELISA – Detection of Specific Proteins  1. Add an antigen for a specific disease  2. Add blood w/ antibody proteins for the antigen  3. Add a second, colored antigen for the same disease  4. If the antibody protein for a disease is there, the well will change color.

Summary  Southern Blotting is a test for genetic disease.  It works by absorbing DNA on an electrophoresis gel onto a membrane  The membrane is then treated with a probe that will bind only to a specific DNA sequence (such as a gene for a genetic disease)  If the gene is present, it will ‘light up’  ELISA is a test for infectious disease  ELISA works by coating wells in a dish with an antigen for a disease  If an individual has the disease, they will have antibodies in their blood for the disease  These antibodies will bind to the antigen; a second antigen will cause a color change in the well if they have the disease.